In recent years, many bio-pharmaceuticals (antibody drugs, biologics, hormones, proteins, etc.) have contributed to medical innovation. However, immunogenicity possessed by these bio-pharmaceuticals is controversial, for example, from the viewpoint of efficacy and safety. In general, a property of an antigen that induces antibody production or cell-mediated immunity is called immunogenicity. The bio-pharmaceuticals can act as antigens to induce antibody production in the bodies of patients. In such a case, neutralizing antibodies against the bio-pharmaceuticals are produced, sometimes resulting in the reduced efficiency of treatment. Alternatively, allergic response, leaching reaction, infusion reaction, or the like may be caused. Alternatively, antibodies that cause autoimmune diseases or the like due to the neutralization of endogenous self-proteins may be produced in response to the bio-pharmaceuticals.
For the process of antibody production, it is important that an antigen is presented on a major histocompatibility complex (also referred to as a MHC molecule) present on the cell surface of an antigen-presenting cell (APC) (this is called “antigen presentation”). A MHC I molecule (class I) and a MHC II molecule (class II) are known as MHC molecules involved in the antigen presentation. For example, the MHC I molecule acts on killer T cells (CD8-positive T cells), and the MHC II molecule acts on helper T cells (CD4-positive T cells). The MHC I molecule acts on endogenous antigens in autologous cells, while the MHC II molecule acts on foreign antigens. Thus, antigen-antibody reaction or the like can be caused, for example, against cancer antigens produced in cancer cells, through antigen presentation mediated by the MHC I molecule. On the other hand, antigen-antibody reaction or the like can be caused against foreign antigens such as the bio-pharmaceuticals, or toxins through antigen presentation mediated by the MHC II molecule.
More specifically, in the case of the event mediated by the MHC I molecule, endogenous proteins in autologous cells are decomposed into smaller peptides by proteasome. Subsequently, each peptide binds to the MHC I molecule synthesized in the vesicle to form a complex. Then, the complex is delivered to the cell surface so that the peptide is presented as an epitope on the MHC I molecule.
On the other hand, in the case of the event mediated by the MHC II molecule, foreign proteins are first taken up into antigen-presenting cells by endocytosis. Subsequently, the taken-up proteins are decomposed into smaller peptides by lysosome. Then, each peptide binds to the MHC II molecule to form a complex. Then, the complex is delivered to the cell surface so that the peptide is presented as an epitope on the MCH II molecule. Subsequently, a T cell receptor on a helper T cell can bind to the antigen-presenting cell via the peptide.
However, these pathways are not definitive, and even foreign antigens may be processed by the MHC I molecule-mediated antigen presentation pathway (this is called “cross-priming”).
In order to circumvent the immunogenicity of antibody drugs, etc., research has been conducted on the identification of peptide sequences presented on MHC molecules. This permits the prediction of the immunogenicity of proteins or peptides intended to be administered to organisms. Furthermore, epitopes can be modified by site-directed mutagenesis for the purpose of producing non-immunogenic proteins, for example, on the basis of information on epitope sequences. A method using a prediction algorithm in silico and T cell proliferation assay (e.g., the measurement of the ability of helper T cells to proliferate by the uptake of tritium-labeled thymidine) are known as methods for identifying peptide sequences. However, it has been difficult to predict an epitope sequence, for example, only from the binding affinity between an epitope candidate peptide and a MHC molecule. Accordingly, there has been a demand for more accurately predicting an epitope that may play a causative role in the induction of immunogenicity by directly identifying the sequence of a peptide presented on a MHC molecule.
Methods have been developed which involve contacting an antigen-presenting cell such as a dendritic cell (DC) with a protein to induce antigen presentation, allowing a MHC molecule on the cell to present a peptide derived from the protein, then separating and purifying a complex of the MHC molecule and the peptide, then eluting the peptide, and directly identifying the sequence of the peptide by use of liquid chromatography mass spectrometry (LC/MS) or the like (Patent Literature 1, Patent Literature 2, and Non Patent Literature 1). These methods are called MAPPs (MHC-associated peptide proteomics).